Homeoprotein signaling in the developing and adult nervous system

Abstract

Signaling classically involves the secretion of diverse molecules that bind specific cell-surface receptors and engage intracellular transduction cascades. Some exceptions-namely, lipophilic agents-can cross plasma membranes to bind intracellular receptors and be carried to the nucleus to regulate transcription. Homeoprotein transcription factors are among the few proteins with such a capacity. Here, we review the signaling activities of homeoproteins in the developing and adult nervous system, with particular emphasis on axon/cell migration and postnatal critical periods of cerebral cortex plasticity. We also describe homeoprotein non-cell-autonomous mechanisms and explore how this "novel" signaling pathway impacts emerging research in brain development and physiology. In this context, we explore hypotheses on the evolution of signaling, the role of homeoproteins as early morphogens, and their therapeutic potential for neurological and psychiatric diseases.

Figure 1

Homeoproteins and boundary formation. A. Examples of boundaries defined by the expression of abutting homeoproteins with self-activating and reciprocal inhibitory properties. B. The classical example of the competitive activities of Emx2 and Pax6 in the definition of primary somato-sensory and visual cortex areas in the developing mouse cortex. The latter work by the group of Denis O’Leary (O’Leary et al., 2007) refers to the cell autonomous activity of homeoproteins and does not consider their non-cell autonomous activity. C. The graded expression of a morphogen creates several domains within the morphogenetic field, according to the Wolpert’s French flag paradigm (Wolpert, 1969). Each domain is characterized by the expression of a homeoprotein transcription factor (Blue-Yellow-Red) but the boundaries are initially fuzzy.

Figure 2

Embryonic and adult expression of Engrailed and Otx2 homeoproteins. At embryonic day E11.5, graded En1/2 expression irradiates from the mid-hindbrain barrier (MHB) into the midbrain. En-1 is also expressed in two lateral stripes along the hindbrain. At E15.5, En-1 expression intensifies around the MHB while En-2 expression extends into the cerebellum. Restricted brain expression of En1/2 continues through to the adult in structures such as the ventral segmental area (VTA), substantia nigra pars compacta, inferior colliculus and the cerebellum. At E11.5, Otx2 expression is restricted to the forebrain and midbrain and stops sharply at the MHB. At E15.5, Otx2 continues to be expressed in restricted midbrain and forebrain structures while patches of expression appear in the cerebellum and hindbrain. Strong Otx2 expression is observed in the choroid plexus (labeled with ‘*’). In the adult, Otx2 is expressed in structures such as the VTA, lateral geniculate nucleus, superior colliculus, medial septum, cerebellum and choroid plexus.

Figure 3

Homeoproteins bind to a wide range of molecules. The homeodomain contains a conserved 3-helix bundle flanked by variable N- and C-terminal domains that together form the homeoprotein. These domains make a plethora of interactions with DNA, RNA, GAGs and proteins. Consequently, homeoproteins are shown to regulate transcription, RNA processing and translation, DNA replication and damage response and are also implicated in cell signaling. Within the 2^nd and 3^rd DNA-binding α-helices are the motifs that permit the non-conventional secretion and internalization of the homeodomain.

Figure 4

The graded expression of a morphogen creates several domains within the morphogenetic field, according to the Wolpert’s French flag paradigm (Wolpert, 1969). Each domain is characterized by the expression of a homeoprotein transcription factor (Blue-Yellow-Red) but the boundaries are initially fuzzy. In this model the further regularity and positioning of the boundary is permitted by the local diffusion of homeoproteins with self-activating and reciprocal inhibitory properties, thus acting as local Turing’s morphogens.

Figure 5

Pax6 defines eye anlagen. A. An extracellular anti-Pax6 antibody present at early developmental stages in the zebrafish does not modify the initial Pax6 expression analyzed by ISH (3 somite stage) but disrupts the development of the eye anlagen (15 somite stage) (Lesaffre et al., 2007). B. The extracellular anti-Pax6 antibody (as in A) leads to a variety of abnormal eye phenotypes (Lesaffre et al., 2007). C. A working hypothesis for the expansion of the eye anlagen includes the requirement for Pax6 transfer between cells.

Figure 6

Graded expression of Engrailed guides retinotectal axons. A. Axons originating from the retina within a temporal-to-nasal EphA2 gradient project onto the tectum within posterior to anterior En1/2 and EphrinA5 gradients. Temporal retinotectal growth cones remain within the anterior tectum while nasal growth cones are attracted to posterior tectum. B. Ectopic expression of En1/2 within anterior tectum results in local repulsion and attraction of temporal and nasal growth cones, respectively (Drescher et al., 1997; Friedman and O’Leary, 1996; Itasaki and Nakamura, 1996; Logan et al., 1996; Shigetani et al., 1997). C. Local expression of secreted single-chain antibody against En1/2 disrupts axon guidance in vivo and results in overgrowth of temporal growth cones that invade the posterior tectum (Wizenmann et al., 2009). D. In vitro guidance assays show that En1/2 attracts nasal growth cones and repels temporal growth cones. This activity is in part local as shown by the attraction and repulsion of nasal and temporal growth cones separated from their cell bodies (Brunet et al., 2005). E. The local activity of En1/2 acts in part through mTOR-dependent translation and results in expression mitochondrial proteins. This activity has local effects on mitochondria, cytoskeleton and other unidentified targets leading to remote effects in the nucleus. En1/2 might also undergo retrograde transport to participate in regulating remote nuclear activity such as transcription and epigenetic events.

Figure 7

Synergy exists between EphrinA5 and En1/2 in local growth cones. A. A consequence of EphrinA5/En1/2 synergistic activity is that En1/2 signaling does not operate in absence of EphrinA5 (knock out) or in excess of EphrinA5 (gain of function) but only at EphrinA5 physiological concentrations. B. ATP signaling is driven by En1/2 through local translation and synergizes with EphrinA5 signaling to elicit growth cone responses (Stettler et al., 2012).

Figure 8

The association between Otx2 and proteoglycans results in cell-specific accumulation. A. Otx2 is attracted to fast-spiking parvalbumin (FSPV) cells through high affinity interactions with chondroitin sulfate (CS) proteoglycans in the perineuronal nets (PNNs) that ensheath these cells (Beurdeley et al., 2012). As polysialic acid is required for infused recombinant Otx2 to diffuse within the parenchyme, we hypothesize that endogenous Otx2 associates with low affinity sugars (heparin sulfate (HS) or HS-proteoglycans) upon secretion from the choroid plexus into the cerebrospinal fluid (CSF). This association could help stabilize Otx2 in the CSF and might facilitate its diffusion in the parenchyme and its specific capture by cells expressing complex sugars with very high Otx2 affinity. B. Alignment of a subset of homeoproteins that contain an RK-, RR-, KR- or KK-doublet within a glycosaminoglycan (GAG) binding motif just upstream of the homeodomain. This motif is implicated in the specificity of Otx2 for the PNNs of FSPV cells and may help establish a sugar code for homeoprotein internalization specificity in vivo.

Figure 9

A two-threshold model for Otx2 in critical period timing. Otx2 accumulates in FSPV-cells after eye opening in the mouse and opens a critical period of heightened plasticity once a first concentration threshold is reached. Continued accumulation leads to a second threshold after which the critical period closes. Several molecules, such as growth factors and receptors, and events, such as excitatory-inhibitory balance and epigenetics, work in concert with Otx2 to “turn” plasticity either on or off. During the critical period, changes in extracellular matrix and cell homeostasis provide a permissive state for the remodeling of connectivity. Afterwards and throughout adulthood, Otx2 continues to bathe the cortex and maintains the cortical network in a mature non-plastic state. By reducing Otx2 accumulation in FSPV cells to levels below the second threshold in the adult, cortical plasticity is induced and opens the possibility of new adult therapies.